Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs (HA-MRSA) by virtue of their genomic background and increased virulence in animal models. Resistance to methicillin in S. aureus is mediated by mecA which is embedded within a mobile genetic element (21-67 kb) called SCCmec. There are five types of SCCmec in MRSA, which differ with respect to the mec complex (mecI-mecR1-mecA), integrated plasmids, transposons, IS elements and other accessory genes. USA300 and MW2 (USA400), the two most common CA-MRSA isolates, carry the SCCmec type IV while USA100 and USA200, which are common HA-MRSAs, contain type II. Recent studies from our lab have shown that a loss of penicillin-binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance in CA-MRSA, but not in HA- MRSA. This finding was confirmed with cefoxitin, a semi-synthetic beta-lactam that binds PBP4 irreversibly, which exhibits synergistic killing with oxacillin against CA-MRSA strains but not against HA-MRSA strains. As all MRSA strains contain mecA, these data indicate that that mecA encoding PBP2A is not the sole determinant of methicillin resistance in CA-MRSA strains. Based on the differences in SCCmec types, we hypothesize that the accessory genes within SCCmec type IV in CA-MRSA strains USA300 and USA400 confer sensitivity to oxacillin (also nafcillin) in the absence of pbp4 or in the presence of cefoxitin. To define the bacterial determinants, we have developed two aims: I) mapping the bacterial determinants that confer sensitivity to oxacillin/nafcillin in the absence of pbp4 or in the presence of PBP4-binding beta-lactam such as cefoxitin;II) characterization of the genetic determinants that confer oxacillin sensitivity in the presence of cefoxitin in CA-MRSA strains USA300 and USA400. With these studies, we will identity the genes in type IV SCCmec responsible for this phenotype (Aim 1). Mutants of these genes in USA300 and USA400 will be constructed and characterized with respect to antibiotic sensitivity, cell wall metabolism and cell wall morphology (Aim II). Upon completion of these studies, we seek to identify genetic determinants that impact oxacillin resistance in CA-MRSA. These genetic determinants may be targets for the development of novel antimicrobial therapy. PUBLIC HEALTH RELEVANCE: Community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) has become a major health problem. Contrary to hospital-acquired methicillin resistant S. aureus, CA-MRSAs are more virulent and can affect healthy individuals. We have found an "Achilles Heel" in CA-MRSA in that penicillin binding protein 4 (PBP4) is required for methicillin resistance in CA-MRSA. There are genes in CA-MRSA involved in this oxacillin sensitivity upon deletion of the pbp4 gene. Our goal is to identify and characterize these genes that confer oxacillin sensitivity.